Neuroinfl ammatory mechanisms in neonatal ischemic stroke based on the bibliographic study are approached in the article. Stroke is a rare disease in children with an estimated incidence between 2 to 13 for 100000 of population and has a signifi cant impact on morbidity and mortality. Ischemic stroke most often occurs during prenatal and fi rst 28 days with a frequency of 1: 4000 live births. Neuroinfl ammation is one of the main mechanisms underlying development of stroke. In this context, it is important to study the infl ammatory markers responsible for the onset and pathogenesis of stroke in children. Among the infl ammatory biomarkers mentioned in presented study are: proinfl ammatory cytokines such as IL-6, IL-1β, but also other biological molecules and factors including vascular endothelial growth factor (VEGF), ciliary neurotrophic factor (CNTF), S100B protein, CD105 endoglin, antiphospholipid antibodies (aPL). Synthesis of literature data shows that ischemic stroke is defi ned by the loss of brain function which is caused by diminished cerebral blood fl ow in the aff ected area. After ischemic stroke is occurred, the blood supply in the area of the aff ected brain tissue is diminished, which leads to oxygen defi ciency in the brain neurons. Angiogenesis occurs in the human brain after the stroke. Ischemia induces a signifi cant increase in microvascular density which is a sign of angiogenesis that occurs after cerebral infarction. Knowing the neuroinfl ammatory mechanisms responsible for development and pathogenesis of neonatal ischemic stroke is important for assessing the infl ammatory responses. Conclusions: Determining the etiology of stroke in children is a very important issue, being caused by multiple etiological factors, diff erent from the adult. Various etiopathogenetic aspects of stroke in children are determined mainly by risk factors. Neuroinfl ammation is the main pathogenetic mechanism underlying the development of stroke in children. Levels in serum of infl ammatory markers responsible for the onset and pathogenesis of stroke is important for assessing infl ammatory responses after stroke in children.
Keywords: neonatal ischemic stroke, children, neuroinfl ammation, etiopathogenesis
Ischemic stroke (IS) most often occurs during the prenatal period and in the first 28 days with a frequency of 1:4000 live newborns . Stroke can occur during pregnancy or immediately after birth, without any marked symptoms [7, 8]. Neonatal IS is very important due to significant complications associated with long-term severe neurological and cognitive deficits, including cerebral palsy, epilepsy, neuropsychological and behavioral disorders.
Neuroinflammation is one of the main mechanisms underlying the occurrence and development of stroke. In this context, it is relevant and important to carry out the review of literature on inflammatory markers responsible for the onset and pathogenesis of stroke in children [8, 13]. Clinical studies and investigations in the field have shown that features of inflammatory responses after stroke in children are different from adults .
Due to rapid growing the volume and number of scientific literature sources in the field of molecular biology, biological markers of IS were remarkable reviewed in recent years. In addition to the role of inflammatory markers in diagnosis and prognosis, many other markers and biological factors were added to the field of interest, including cytokines derived from tissues, molecules similar to growth factor, hormones and micro RNA . So far, biomarkers represent a possible challenge in diagnosing and predicting the prognosis of appearance of pathology, pathogenesis and recovery in stroke. Many substances are still being investigated and its role as biological markers can become promising and encouraging. Experimental and clinical research should increase this spectrum and promote new discoveries in this area in order to improve the diagnosis and treatment of pediatric stroke .
Data from literature on molecular biology highlights the major role of biomarkers in diagnosing and evaluating of neurologic prognosis, as well as in elucidating the pathogenesis and recovery in children with stroke. In the list of biomarkers are included some inflammatory markers, i. e., proinflammatory cytokines, such as IL-6, IL-1β, together with other substances and biological factors, including endothelial growth factor (VEGF), ciliary neurotrophic factor (CNTF), S100B protein, CD105 endoglin, antiphospholipid antibodies (aPL), etc. [1, 10, 13,14].
In addition to the role of inflammatory markers, such as IL-6, TNF-A or IL-1β, in diagnosis and prognosis, many other substances and biological factors in the serum or plasma have been reviewed as candidate markers, as cytokines, i. e., miocine, adipokine, substances related to growth factor, hormones and micro RNA. Neuroinflammation represents the main mechanism underlying the occurrence and development of stroke, and level of soluble immune factors and immune cells can be used as evidence of the occurrence and pathogenesis of stroke as well as recovery process [6-8].
Endoglin (ENG, also known as CD105) is a receptor associated with tumor growth factor β (TGFβ), which is necessary for vasculogenesis as well as angiogenesis . Angiogenesis is important for cerebral vascularization and for the pathogenesis of cerebral vascular diseases. ENG is an essential component of the activation complex of nitric oxide by endothelium. Animal studies have shown that ENG deficiency have an impact on the recovery after the stroke. ENG deficiency also affects the regulation of vascular tonus, which has a contribution in the pathogenesis of cerebral vascular anomalies and vascular spasm .
Choi E. J., Walker E. J. et al. showed that ENG was very expressed in the ischemic zone of human stroke, where an increase of angiogenesis was found . The role of ENG in stroke is extremely complex. Thus, exaggerated ENG expression is amplify the signaling of tumor growth factor (TGFβ) and promotes the reshaping of the new vascular wall . ENG overexpression also protects endothelial cells against apoptosis induced by TGFp. Reducing vascular cell apoptosis after hypoxia improves blood intake to ischemic tissue. Increasing ENG expression in endothelial cells could also be dangerous because the permeability of the blood brain barrier can be increased in some of the capillaries where the level of ENG expression is high, being accompanied by the infiltration of mononuclear cells in adjacent cerebral tissue . These findings suggest that ENG overexpression could affect the integrity of the vessel’s wall. At the same time, the lack of ENG expression may indicate severe vascular damage . ENG is involved in the pathogenesis of post-ischemic cerebral lesions in humans. ENG abnormality could lead to longterm neurological deterioration or the emergence of cognitive disorders after an acute IS .
Recently vascular endothelial growth factor (VEGF) was associated with Brain-derived neurotrophic factor (BDNF) as biomarker of stroke despite some controversies . In animal models, ciliary neurotrophic factor (CNTF), which is endogenously regulated during a stroke onset, mediates neurogenesis and anti-inflammatory processes . So far there is no evidence of the role of serum CNTF in stroke, although the levels of this neurotrophine in peripheric blood are much more important for the patients with amyotrophic lateral sclerosis . Vascular endothelial growth factor (VEGF) have been shown to play role in atherosclerosis, arteriogenesis, cerebral edema, neuroprotection, neurogenesis, angiogenesis, postischemic events with subsequent repair of vessels, having an impact on the effects of transplanted stem cells in experimental stroke. Most of these processes involve VEGF and its receptor, VEGFR-2. Thus, VEGF-B, the placental growth factor and the VEGFR-1 were involved only in some cases.
VEGF signalling pathways represents potential objectives for treatment of stroke in acute and chronic stages [4, 5]. VEGF-A is a primary mediator of cerebral angiogenesis, which levels have elevated after stroke in rodents and humans . Brain angiogenesis is a wellcontrolled process, being regulated by derivatives on neuroectoderm that bind to tyrosin kinase receptors expressed on endothelial cells . In the brain of rat angiogenesis is complete approximately at the age of 20 days. However, in the adult brain under pathological conditions such as hypoxia/ischemia and growth of brain tumor, endothelial cells may proliferate. Current evidence suggests that physiological angiogenesis in the brain is regulated by similar mechanisms, as in pathological angiogenesis induced by tumors or hypoxia/ischemia. Cellular endothelial mitogen is activated in hypoxia, and vascular permeability factor or VEGF seems to play a key role in most of these processes . VEGF is expressed when angiogenesis is increased, as in embryonic neuroectoderm, in glioblastomas and in myocardial infarction. However, the expression is decreased in the absence of angiogenesis, as well as in neuroectoderm in adults. On the other hand, the induction of angiogenesis by growth factors, i. e., proangiogenesis, may prove to be an effective therapy in patients with stroke [4, 5].
S100B protein is the best studied biomarker in stroke. It possesses intracellular and extracellular properties. Intracellular, S100B plays role in homeostasis of calcium, thus transferring signals from secondary messengers . This protein is involved in cell differentiation and the progression of the cellular cycle having the effect of inhibiting apoptosis if applied under experimental conditions. Extracellular, both in normal physiology and in traumatic conditions, S100B protein stimulates neurogenesis and neural plasticity, has neuromodulation effect and intensifies the processes involved in memory and learning . Was showed that extracellular increasing the level of S100B protein lead to neural dysfunction or to the death of cells due to an inflammatory response that stimulates astrocytes and microglia, as for these processes produce proinflammatory cytokines, with subsequent increasing the extracellular levels of calcium and activation of nitric oxide with harmful effects. Some studies emphasize different effects of S100B protein that depend on the receptor for advanced endoglycan products, which is regulated by the elevated levels of the S100B protein and which can cause the activation of the proinflammatory genes [1, 2].
The synthesis of literature data shows that IS is defined by deterioration of cerebral function caused by diminishing cerebral blood flow in the affected area. After this accident occurs, the blood intake to the affected brain tissue is reduced, leading to oxygen deficiency in the brain cells. Angiogenesis occurs in the human brain after the stroke. Ischemia induces a significant increase in microvascular density, a sign of angiogenesis, in the focus of cerebral infarction [3,14]. The increase in the density of vessels in the ischemic focus is positively correlated with the survival rate of patients with stroke . In addition, increased angiogenesis was associated with improving functional results both in animal models and in patients with stroke .
Clémence Guiraut et al. showed that after the stroke increases cytokine levels as a result of increasing the production of inflammatory, glial and neural cells with IL-1, IL-6, IL-10, tumour necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β), which were best studied in stroke . IL-1β and TNF-α were associated with exacerbation of lesions in stroke, while it was found that IL-6, IL-10 and TGF-β have neuroprotective effects  (fig. 1).
Inflammation and coagulation are closely related processes in mutual manner. For example, thrombin, which is an activator of the coagulation cascade, is also able to induce the expression of cytokines and proinflammatory chemokines by endothelial cells. Platelets involved not only in clotting to protect the body from damage of arterial wall but also in the capture and elimination of bacterial agents. Therefore, the thrombotic process is associated with the migration to the lesion site of the immune cells, which process can be called also as “immunothrombotic” . Activated platelets have an important role in immunothrombosis, as it is contribute to the migration of neutrophils and monocytes, their adhesion and activation induces proinflammatory response. These immune cells could induce devastating inflammation within the wall of a thrombosis artery, which would trigger a subsequent occlusion and thrombosis, as observed in intravascular coagulation disseminated after sepsis. Therefore, the imunotrombotice processes occurring inside the lumen and inflammatory processes could be part of a vicious circle that reveals the pathophysiology of neonatal ischemic strokes, even in a primary embolic process that it triggers the inflammation of the arterial wall.
Based on above mentioned clinical and preclinical findings, neonatal ischemic stroke results from mechanisms combining perinatal inflammation and hypoxic ischemia (HI). In contrast to HI or postnatal inflammation occurring at least 24 hours before HI, preclinical studies showed a neuroprotective role of prenatal inflammation which occurs immediately after to postnatal HI lesions . In newborns, such prenatal inflammatory exposure, e. g., caused by corionamnionitis, could be involved in the worsening of HI lesions. Inflammatory pathways involved in HI and in infection or inflammation which is characteristic for neonatal ischemic stroke are given below.
The first phase of the lesion in neonatal IS occurs between 0 and 6 hours after starting of hypoxicischemic (HI) event or infection/inflammation accompanied by HI . This phase is characterized by different types of cellular death, including necrosis and necroptosis. Primary cell lesions will induce the activation of several inflammatory cascades. Exposure to lipopolysaccharide (LPS) and HI leads to the activation of various cellular response mechanisms inside the neurons, leading to an overexpression of IL-1 β, to the synthesis of tumor necrosis factor (TBF-α) induced by the nuclear factor kappa B (NFκB), activating inflammasomes. These phenomena will lead to the activation of glial cells and increased inflammation by releasing reactive oxygen species (ROS) and several inflammatory substances . The neural cellular stress associated with ischemic stroke is mainly due to the combination of energy insufficiency, the excess of intracellular Ca2+ ions and release of glutamate, as well as ionic imbalance and oxidative stress. All these mechanisms determine the death of neurons. Cellular death, necrosis and necroptosis occur between 0 and 6 hours after occurring of HI or other pathogenetic mechanisms in combination with HI.
Antoine Giraud et al. showed that exposure to lipopolysaccharides and HI leads to the formation of a vicious cycle in which occurs the neural selfdestruction through the mediators of inflammation, i. e., IL-1β, TNF-α, which show the production of reactive oxygen spesies (ROS) and apoptosis induced by mitogen-activated protein kinase (MAPK) . Lipopolysaccharides combined with HI lead to the activation of glia and synthesis of neurotoxic substances, such as matrix metalloproteinase induced by IL-1β, nitric oxide (NO) and inducible nitric oxide synthase (iNOS). When the brain is exposed to lipopolysaccharides and HI, NO and IL-1β potentiates effect and contributes to the destruction of the blood-brain barrier (BBB) due to degradation of lamina of intracerebral vessels. As a result of this injury, proinflammatory agents and/or neurotoxins can penetrate freely through BBE. Necroptosis represents early cellular death and is triggered by the inflammatory TNF-α mediators and tumor necrosis factor receptor superfamily (TNFR), i. e., TNFR1, FAS and TLR (mostly TLR-3 and TLR-4). Necrosome is a complex that requires the presence of activated receptor that interacts with receptorinteracting protein kinases, i. e., RIP-1, RIP-3, and pseudo-kinase, i. e., MLKL, to carry out necroptosis. All the mechanisms by which MLKL induce cellular death are not completely elucidated. Was showed fact that cerebral expression of TNF-α is triggered by exposing of lipopolysaccharides and HI .
The secondary phase occurs between 24 and 72 hours after neonatal IS and includes processes such as cell apoptosis, anoikis and autophagy. As a whole, these processes will induce activation of cerebral vascular endothelium and probably damage of blood brain barrier and infiltration of leukocytes into the brain. Preclinical studies have shown that apoptotic mechanisms have a neuroprotective role  (fig. 2). It is known that the mechanism of cellular death by apoptosis and autophagy intersects and that autophagy can block apoptosis by blockage of mitochondria. Inducing of autophagy immediately after neonatal HI can serve as a neuroprotective mechanism that will limit apoptosis. On the other hand, autophagy appears to be involved in the cell death induced by HI. In addition, another form of apoptosis, i. e., anoikis, could develop after HI and/ or inflammation .
The establishment of stroke etiology in children is a very important area, implicating multiple etiologic factors, which are different from the adult. Various etiopathogenic aspects of stroke in children are mainly related to risk factors. Neuroinflammation constitutes the main pathogenetic mechanism underlying stroke development in children. Serum level of inflammatory markers responsible for the onset and pathogenesis of stroke is important for the assessment of inflammatory responses occurring after a stroke in children.
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